Pilus proteins and compositions

Abstract

The invention provides methods of forming pili in vitro and proteins suitable for use in these methods. The invention also provides pili produced by these methods and compositions comprising these pili for the treatment and prevention of bacterial disease, in particular of conditions caused by Streptococcus.

1 . A method of ligating at least two moieties comprising contacting the at least two moieties with a pilus-related sortase C enzyme in vitro under conditions suitable for a sortase mediated transpeptidation reaction to occur, wherein the pilus-related sortase C enzyme comprises an exposed active site. 
     
     
         2 . The method of  claim 1 , wherein the pilus-related sortase C enzyme comprises an amino acid sequence having at least 60% identity to or at least 50 consecutive amino acids of a sortase C polypeptide from  Streptococcus.    
     
     
         3 . The method of  claim 2  wherein the  Streptococcus  is selected from the group consisting of  Streptococcus agalactiae  (GBS),  Streptococcus pneumonia  (pneumococcus) and  Streptococcus pyogenes  (GAS). 
     
     
         4 . The method of  claim 1 , wherein the pilus-related sortase C enzyme is a sortase C1 enzyme (srtC1), sortase C2 enzyme (SrtC2), a sortase C3 enzyme (SrtC3), or combination thereof. 
     
     
         5 . The method of  claim 1 , wherein the pilus-related sortase C enzyme comprises a deletion of part or all of the lid region of the sortase C enzyme. 
     
     
         6 . The method of  claim 1 , wherein the pilus-related sortase C enzyme comprises a mutation at positions 84, 85 and/or 86 of the amino acid sequence of the GBS sortase C1 enzyme of PI-2a (SEQ ID NO:3), or at corresponding positions in the amino acid sequence of another pilus-related sortase C enzyme, wherein the mutation is a deletion, substitution, or combination thereof. 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the pilus-related sortase C enzyme comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 and 71. 
     
     
         9 . The method of  claim 1 , wherein the at least two moieties comprise an LPxTG motif, a pilin motif, an E-Box motif, or combination thereof. 
     
     
         10 . The method of  claim 1 , wherein the at least two moieties correspond to polypeptides of Gram-positive bacteria. 
     
     
         11 . (canceled) 
     
     
         12 . The method of  claim 10 , wherein the at least two moieties are polypeptides having 50% or more identity to or a fragment with at least 20 consecutive amino acids of Streptococcal backbone proteins and/or ancillary proteins. 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 12 , wherein the at least two moieties comprise or consist of an amino acid sequence: (a) having 50% or more identity to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, or 97; or (b) that is a fragment of at least ‘n’ consecutive amino acids of one of these sequences wherein ‘n’ is 20 or more. 
     
     
         15 . An artificial pilus obtained or obtainable by the method of  claim 1 . 
     
     
         16 . An artificial pilus which comprises at least two variants of backbone protein GBS59 and wherein the at least two variants are selected from the group consisting of Group B  Streptococcus  strains 2603, H36B, 515, CJB111, CJB110 and DK21. 
     
     
         17 - 18 . (canceled) 
     
     
         19 . A method of treating or preventing Streptococcal infection in a patient in need thereof comprising administering to the patient the artificial pilus of  claim 15  or  16  in an amount effective to treat or prevent the Streptococcal infection. 
     
     
         20 . The method of  claim 1 , wherein the at least two moieties comprise a first moiety comprising the amino acid motif LPXTG, wherein X is any amino acid, and a second moiety comprising at least one amino acid. 
     
     
         21 - 25 . (canceled) 
     
     
         26 . The method of  claim 20 , wherein either the first moiety or the second moiety comprises a detectable label. 
     
     
         27 . (canceled) 
     
     
         28 . The method according to  claim 20 , wherein either the first moiety or the second moiety is:
 i) a polypeptide and the other moiety is a protein or glycoprotein on the surface of a cell;   ii) a polypeptide and the other moiety comprises amino acids conjugated to a solid support; or,   iii) a polypeptide and the other moiety comprises at least one amino acid conjugated to a polynucleotide.   
     
     
         29 - 30 . (canceled) 
     
     
         31 . The method of  claim 20 , wherein the first moiety and the second moiety are the N-terminus and C-terminus of a polypeptide chain, and ligation results in the formation of a circular polypeptide. 
     
     
         32 . (canceled) 
     
     
         33 . A kit comprising a PI-2b sortase C1 or a PI-2b sortase C2 enzyme from  Streptococcus agalactiae  and a moiety comprising the amino acid motif LPXTG, wherein X is any amino acid. 
     
     
         34 . A conjugate obtained or obtainable by the method of  claim 20 .